Characterization of the Nonendocrine Cell Populations in Human Embryonic Stem Cell-Derived (hESC) Islet-Like Clusters Posttransplantation.

Islet-like clusters derived from human embryonic stem cells (hESC) hold the potential to cure type 1 diabetes mellitus. Differentiation protocols of islet-like clusters lead to the generation of minor fractions of nonendocrine cells, which are mainly from endodermal and mesodermal lineages, and the risk of implanting these is unclear. In the present study, the histogenesis and the tumorigenicity of nonendocrine cells were investigated in vivo. Immunodeficient mice were implanted under the kidney capsule with islet-like clusters which were derived from differentiation of cells batches with either an intermediate or poor cell purity and followed for 8 or 26 weeks. Using immunohistochemistry and other techniques, it was found that the intermediate differentiated cell implants had limited numbers of small duct-like cysts and nonpancreatic tissue resembling gastrointestinal and retinal pigmented epithelium. In contrast, highly proliferative cystic teratomas were found at a high incidence at the implant site after 8 weeks, only in the animals implanted with the poorly differentiated cells. These findings indicate that the risk for teratoma formation and the amount of nonpancreatic tissue can be minimized by careful in-process characterization of the cells and thus highlights the importance of high purity at transplantation and a thorough ex-vivo characterization during cell product development.

Partial Disruption of Lipolysis Increases Postexercise Insulin Sensitivity in Skeletal Muscle Despite Accumulation of DAG.

Type 2 diabetes and skeletal muscle insulin resistance have been linked to accumulation of the intramyocellular lipid-intermediate diacylglycerol (DAG). However, recent animal and human studies have questioned such an association. Given that DAG appears in different stereoisomers and has different reactivity in vitro, we investigated whether the described function of DAGs as mediators of lipid-induced insulin resistance was dependent on the different DAG isomers. We measured insulin-stimulated glucose uptake in hormone-sensitive lipase (HSL) knockout (KO) mice after treadmill exercise to stimulate the accumulation of DAGs in skeletal muscle. We found that, despite an increased DAG content in muscle after exercise in HSL KO mice, the HSL KO mice showed a higher insulin-stimulated glucose uptake postexercise compared with wild-type mice. Further analysis of the chemical structure and cellular localization of DAG in skeletal muscle revealed that HSL KO mice accumulated sn-1,3 DAG and not sn-1,2 DAG. Accordingly, these results highlight the importance of taking the chemical structure and cellular localization of DAG into account when evaluating the role of DAG in lipid-induced insulin resistance in skeletal muscle and that the accumulation of sn-1,3 DAG originating from lipolysis does not inhibit insulin-stimulated glucose uptake.

Contraction-induced lipolysis is not impaired by inhibition of hormone-sensitive lipase in skeletal muscle.

In skeletal muscle hormone-sensitive lipase (HSL) has long been accepted to be the principal enzyme responsible for lipolysis of intramyocellular triacylglycerol (IMTG) during contractions. However, this notion is based on in vitro lipase activity data, which may not reflect the in vivo lipolytic activity. We investigated lipolysis of IMTG in soleus muscles electrically stimulated to contract ex vivo during acute pharmacological inhibition of HSL in rat muscles and in muscles from HSL knockout (HSL-KO) mice. Measurements of IMTG are complicated by the presence of adipocytes located between the muscle fibres. To circumvent the problem with this contamination we analysed intramyocellular lipid droplet content histochemically. At maximal inhibition of HSL in rat muscles, contraction-induced breakdown of IMTG was identical to that seen in control muscles (P < 0.001). In response to contractions IMTG staining decreased significantly in both HSL-KO and WT muscles (P < 0.05). In vitro TG hydrolase activity data revealed that adipose triglyceride lipase (ATGL) and HSL collectively account for ∼98% of the TG hydrolase activity in mouse skeletal muscle, other TG lipases accordingly being of negligible importance for lipolysis of IMTG. The present study is the first to demonstrate that contraction-induced lipolysis of IMTG occurs in the absence of HSL activity in rat and mouse skeletal muscle. Furthermore, the results suggest that ATGL is activated and plays a major role in lipolysis of IMTG during muscle contractions.

Interleukin-18 activates skeletal muscle AMPK and reduces weight gain and insulin resistance in mice.

Circulating interleukin (IL)-18 is elevated in obesity, but paradoxically causes hypophagia. We hypothesized that IL-18 may attenuate high-fat diet (HFD)-induced insulin resistance by activating AMP-activated protein kinase (AMPK). We studied mice with a global deletion of the α-isoform of the IL-18 receptor (IL-18R(-/-)) fed a standard chow or HFD. We next performed gain-of-function experiments in skeletal muscle, in vitro, ex vivo, and in vivo. We show that IL-18 is implicated in metabolic homeostasis, inflammation, and insulin resistance via mechanisms involving the activation of AMPK in skeletal muscle. IL-18R(-/-) mice display increased weight gain, ectopic lipid deposition, inflammation, and reduced AMPK signaling in skeletal muscle. Treating myotubes or skeletal muscle strips with IL-18 activated AMPK and increased fat oxidation. Moreover, in vivo electroporation of IL-18 into skeletal muscle activated AMPK and concomitantly inhibited HFD-induced weight gain. In summary, IL-18 enhances AMPK signaling and lipid oxidation in skeletal muscle implicating IL-18 in metabolic homeostasis.

AMPK and insulin action--responses to ageing and high fat diet.

The 5'-AMP-activated protein kinase (AMPK) is considered "a metabolic master-switch" in skeletal muscle reducing ATP- consuming processes whilst stimulating ATP regeneration. Within recent years, AMPK has also been proposed as a potential target to attenuate insulin resistance, although the exact role of AMPK is not well understood. Here we hypothesized that mice lacking α2AMPK activity in muscle would be more susceptible to develop insulin resistance associated with ageing alone or in combination with high fat diet. Young (∼4 month) or old (∼18 month) wild type and muscle specific α2AMPK kinase-dead mice on chow diet as well as old mice on 17 weeks of high fat diet were studied for whole body glucose homeostasis (OGTT, ITT and HOMA-IR), insulin signaling and insulin-stimulated glucose uptake in muscle. We demonstrate that high fat diet in old mice results in impaired glucose homeostasis and insulin stimulated glucose uptake in both the soleus and extensor digitorum longus muscle, coinciding with reduced insulin signaling at the level of Akt (pSer473 and pThr308), TBC1D1 (pThr590) and TBC1D4 (pThr642). In contrast to our hypothesis, the impact of ageing and high fat diet on insulin action was not worsened in mice lacking functional α2AMPK in muscle. It is concluded that α2AMPK deficiency in mouse skeletal muscle does not cause muscle insulin resistance in young and old mice and does not exacerbate obesity-induced insulin resistance in old mice suggesting that decreased α2AMPK activity does not increase susceptibility for insulin resistance in skeletal muscle.

Synthesis and characterization of nanogels of poly(N-isopropylacrylamide) by a combination of light and small-angle X-ray scattering.

Thermo-responsive crosslinked nanogels of N-isopropylacrylamide (NIPAM) were synthesized by emulsion polymerization and the size was varied using different concentrations of surfactant (sodium dodecyl sulfate, SDS) in the polymerization process. The collapse behavior of the nanogels at the lower critical solution temperature at around 32 °C was investigated by dynamic light scattering, and by combined static light scattering (SLS) and small-angle X-ray scattering (SAXS). The combined data from SLS and SAXS were analyzed by a model for the nanogels which at intermediate temperatures included a central core and a more diffuse outer layer describing pending polymer chains with a low degree of cross linking. In the expanded state, the particles were modeled with a single component with a broad graded surface. In the collapsed state the nanogels were modeled as homogeneous and relatively compact particles. The amount of surfactant used had a profound effect on the final size of the nanogels owing to the phenomenon of colloidal stabilization of the emulsion droplets during polymerization. The combination of SLS and SAXS as applied to the nanogels is an attractive method for particle characterization as it spans a very large range of scattering vector from q = 0.0004 to 0.22 Å(-1).

Adipose triglyceride lipase plays a key role in the supply of the working muscle with fatty acids.

FAs are mobilized from triglyceride (TG) stores during exercise to supply the working muscle with energy. Mice deficient for adipose triglyceride lipase (ATGL-ko) exhibit defective lipolysis and accumulate TG in adipose tissue and muscle, suggesting that ATGL deficiency affects energy availability and substrate utilization in working muscle. In this study, we investigated the effect of moderate treadmill exercise on blood energy metabolites and liver glycogen stores in mice lacking ATGL. Because ATGL-ko mice exhibit massive accumulation of TG in the heart and cardiomyopathy, we also investigated a mouse model lacking ATGL in all tissues except cardiac muscle (ATGL-ko/CM). In contrast to ATGL-ko mice, these mice did not accumulate TG in the heart and had normal life expectancy. Exercise experiments revealed that ATGL-ko and ATGL-ko/CM mice are unable to increase circulating FA levels during exercise. The reduced availability of FA for energy conversion led to rapid depletion of liver glycogen stores and hypoglycemia. Together, our studies suggest that ATGL-ko mice cannot adjust circulating FA levels to the increased energy requirements of the working muscle, resulting in an increased use of carbohydrates for energy conversion. Thus, ATGL activity is required for proper energy supply of the skeletal muscle during exercise.

A Ca(2+)-calmodulin-eEF2K-eEF2 signalling cascade, but not AMPK, contributes to the suppression of skeletal muscle protein synthesis during contractions.

Skeletal muscle protein synthesis rate decreases during contractions but the underlying regulatory mechanisms are poorly understood. It was hypothesized that there would be a coordinated regulation of eukaryotic elongation factor 2 (eEF2) and eukaryotic initiation factor 4E-binding protein 1 (4EBP1) phosphorylation by signalling cascades downstream of rises in intracellular [Ca(2+)] and decreased energy charge via AMP-activated protein kinase (AMPK) in contracting skeletal muscle. When fast-twitch skeletal muscles were contracted ex vivo using different protocols, the suppression of protein synthesis correlated more closely with changes in eEF2 than 4EBP1 phosphorylation. Using a combination of Ca(2+) release agents and ATPase inhibitors it was shown that the 60-70% suppression of fast-twitch skeletal muscle protein synthesis during contraction was equally distributed between Ca(2+) and energy turnover-related mechanisms. Furthermore, eEF2 kinase (eEF2K) inhibition completely blunted increases in eEF2 phosphorylation and partially blunted (i.e. 30-40%) the suppression of protein synthesis during contractions. The 3- to 5-fold increase in skeletal muscle eEF2 phosphorylation during contractions in situ was rapid and sustained and restricted to working muscle. The increase in eEF2 phosphorylation and eEF2K activation were downstream of Ca(2+)-calmodulin (CaM) but not other putative activating factors such as a fall in intracellular pH or phosphorylation by protein kinases. Furthermore, blunted protein synthesis and 4EBP1 dephosphorylation were unrelated to AMPK activity during contractions, which was exemplified by normal blunting of protein synthesis during contractions in muscles overexpressing kinase-dead AMPK. In summary, in fast-twitch skeletal muscle, the inhibition of eEF2 activity by phosphorylation downstream of Ca(2+)-CaM-eEF2K signalling partially contributes to the suppression of protein synthesis during exercise/contractions.

Adipose triglyceride lipase in human skeletal muscle is upregulated by exercise training.

Mobilization of fatty acids from stored triacylglycerol (TG) in adipose tissue and skeletal muscle [intramyocellular triacylglycerol (IMTG)] requires activity of lipases. Although exercise training increases the lipolytic capacity of skeletal muscle, the expression of hormone-sensitive lipase (HSL) is not changed. Recently, adipose triglyceride lipase (ATGL) was identified as a TG-specific lipase in various rodent tissues. To investigate whether human skeletal muscle ATGL protein is regulated by endurance exercise training, 10 healthy young men completed 8 wk of supervised endurance exercise training. Western blotting analysis on lysates of skeletal muscle biopsy samples revealed that exercise training induced a twofold increase in skeletal muscle ATGL protein content. In contrast to ATGL, expression of comparative gene identification 58 (CGI-58), the activating protein of ATGL, and HSL protein was not significantly changed after the training period. The IMTG concentration was significantly decreased by 28% at termination of the training program compared with before. HSL-phoshorylation at Ser(660) was increased, HSL-Ser(659) phosporylation was unchanged, and HSL-phoshorylation at Ser(565) was decreased altogether, indicating an enhanced basal activity of this lipase. No change was found in the expression of diacylglycerol acyl transferase 1 (DGAT1) after training. Inhibition of HSL with a monospecific, small molecule inhibitor (76-0079) and stimulation of ATGL with CGI-58 revealed that significant ATGL activity is present in human skeletal muscle. These results suggest that ATGL in addition to HSL may be important for human skeletal muscle lipolysis.

Protein-containing nutrient supplementation following strength training enhances the effect on muscle mass, strength, and bone formation in postmenopausal women.

We evaluated the response of various muscle and bone adaptation parameters with 24 wk of strength training in healthy, early postmenopausal women when a nutrient supplement (protein, carbohydrate, calcium, and vitamin D) or a placebo supplement (a minimum of energy) was ingested immediately following each training session. At inclusion, each woman was randomly and double-blindedly assigned to a nutrient group or a placebo (control) group. Muscle hypertrophy was evaluated from biopsies, MRI, and dual-energy X-ray absorptiometry (DEXA) scans, and muscle strength was determined in a dynamometer. Bone mineral density (BMD) was measured using DEXA scans, and bone turnover was determined from serum osteocalcin and collagen type I cross-linked carboxyl terminal peptide. The nutrient group improved concentric and isokinetic (60 degrees /s) muscle strength from 6 to 24 wk by 9 +/- 3% (P < 0.01), whereas controls showed no change (1 +/- 2%, P > 0.05). Only the nutrient group improved lean body mass (P < 0.05) over the 24 wk. BMD responded similarly at the lumbar spine but changed differently in the two groups at the femoral neck (P < 0.05) [control: 0.943 +/- 0.028 to 0.930 +/- 0.024 g/mm(3) (-1.0 +/- 1.4%); nutrient group: 0.953 +/- 0.051 to 0.978 +/- 0.043 g/mm(3) (3.8 +/- 3.4%)] when adjusted for age, body mass index, and BMD at inclusion. Bone formation displayed an interaction (P < 0.05), mainly caused by increased osteocalcin at 24 wk in the nutrient group. In conclusion, we report that nutrient supplementation results in superior improvements in muscle mass, muscle strength, femoral neck BMD, and bone formation during 24 wk of strength training. The observed differences following such a short intervention emphasize the significance of postexercise nutrient supply on musculoskeletal maintenance.

Regulation and function of Ca2+-calmodulin-dependent protein kinase II of fast-twitch rat skeletal muscle.

The activation and function of Ca(2+)-calmodulin-dependent kinase II (CaMKII) in contracting rat skeletal muscle was examined. The increase in autonomous activity and phosphorylation at Thr(287) of CaMKII of gastrocnemius muscle in response to contractions in situ was rapid and transient, peaking at 1-3 min, but reversed after 30 min of contractions. There was a positive correlation between CaMKII phosphorylation at Thr(287) and autonomous CaMKII activity. In contrast to the rapid and transient increase in autonomous CaMKII activity, the phosphorylation of the putative CaMKII substrate trisk95/triadin was rapid and sustained during contractions. There were no changes in CaMKII activity and phosphorylation or trisk95 phosphorylation in the resting contralateral muscles during stimulation. When fast-twitch muscles were contracted ex vivo, CaMKII inhibition resulted in a greater magnitude of fatigue as well as blunted CaMKII and trisk95 phosphorylation, identifying trisk95 as a physiological CaMKII substrate. In summary, skeletal muscle CaMKII activation was rapid and sustained during exercise/contraction and is mediated by factors within the contracting muscle, probably through allosteric activation via Ca(2+)-CaM. CaMKII may signal through trisk95 to modulate Ca(2+) release in fast-twitch rat skeletal muscle during exercise/contraction.